The molecular mechanisms of ion-channel regulation are not well understood. We use Paramecium as a model to study these mechanisms. At least seven different types of ion currents including one Ca-current and three K-currents have been analyzed. We will continue to study the macroscopic activities with the two electrode voltage clamp and the microscopic activities with the patch clamp. We will focus on the Ca-channel and the Ca-dependent K channel because the complementing biochemical studies have led to the discovery of soluble proteins which restore the missing currents in mutants upon microinjections. Membranes of the existing mutants, mutants to be isolated, suppressors and heterozygotes will be examined. Based on open probability, open- and shut-time distributions and other parameters from the patch-clamp experiments, we will construct kinetic models for the wild-type channels and examine the mutant activities in light of these models. Channels in patches from the wild type or mutants will be exposed to various curing factors, enzymes, inhibitors, second messengers and our monoclonal antibodies to calmodulin to further understand the chemistry of channel regulation. The unique combination of mutations, protein factors, voltage clamp, patch clamp, antibodies and microinjection provides a window to study the mechanism of ion-channel function and regulation not open in other biological systems.